This chapter describes the ways that a systematic use of phages led to the development of novel cloning vectors and ultimately transformation of mycobacteria. In addition, it describes how the detailed characterization of one phage, L5, has provided an abundance of novel insights into and genetic tools for mycobacterial molecular genetics. Lastly, it also describes how the combination of a reporter gene with a mycobacteriophage has breathed new life into rapid assessment of drug susceptibilities in clinical samples of Mycobacterium tuberculosis and is also providing a useful tool for screening novel antituberculosis compounds. Mycobacteriophage L5 is the best characterized of the mycobacteriophages. L5 lysogens of M. smegmatis contain a copy of the L5 prophage integrated site specifically into the bacterial chromosome. The introduction of the BACTEC system for clinical analysis of M. tuberculosis has considerably shortened the time needed for drug susceptibility determination. Luciferase reporter mycobacteriophages have the potential of greatly reducing the time needed for M. tuberculosis analysis in a simple and relatively inexpensive assay. The attractions in using luciferase reporter phages for drug screening are many. First, they can provide a readout of results in as little as 2 h. Second, they can be easily adapted to an automated system that uses microtiter plates. The finding of even a single new antituberculosis drug could have an important impact on the control of tuberculosis, and the authors believe that the search for such a drug warrants efforts to develop luciferase reporter antibiotic screening systems for M. tuberculosis.

Contour-clamped homogeneous electric field (CHEF) analysis of mycobacteriophage genomes. A set of mycobacteriophage genomes were analyzed using the CHEF electrophoresis system. Genomes that have cohesive ends tend to form ladders, whereas phages that are terminally redundant or have irregular ends yield single bands. Thus, 13 and Bxbl appear to have irregular ends, while the rest of the phages have cohesive ends.

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Figure 1.

Contour-clamped homogeneous electric field (CHEF) analysis of mycobacteriophage genomes. A set of mycobacteriophage genomes were analyzed using the CHEF electrophoresis system. Genomes that have cohesive ends tend to form ladders, whereas phages that are terminally redundant or have irregular ends yield single bands. Thus, 13 and Bxbl appear to have irregular ends, while the rest of the phages have cohesive ends.

Schematic of shuttle phasmid transfer. Shuttle phasmids have pleiotropic properties. First, they are cosmids, and they can be packaged into λ heads and can replicate in E. coli as cosmids. Alternatively, the shuttle phasmid DNAs can be transfected into M. smegmatis, where they express mycobacteriophage genes and undergo lytic growth. These mycobacteriophage-packaged DNAs can be efficiently introduced into a broad range of mycobacteria, including the slow-growing mycobacteria (reprinted from Jacobs et al., 1989).

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Figure 2.

Schematic of shuttle phasmid transfer. Shuttle phasmids have pleiotropic properties. First, they are cosmids, and they can be packaged into λ heads and can replicate in E. coli as cosmids. Alternatively, the shuttle phasmid DNAs can be transfected into M. smegmatis, where they express mycobacteriophage genes and undergo lytic growth. These mycobacteriophage-packaged DNAs can be efficiently introduced into a broad range of mycobacteria, including the slow-growing mycobacteria (reprinted from Jacobs et al., 1989).

Map of the phage L5 genome. The 52,297-bp genome of L5 is shown as the horizontal bar, with vertical bars spaced 1 kb apart; the attachment site (attP) is located near the center of the genome. The shaded boxes represent the putative L5 genes, with those above the genome being transcribed rightward and those below the genome being transcribed leftward; the different shadings and vertical heights depict the reading frame for each gene. The functions of some of the genes are indicated. At the bottom, the locations of putative promoters are shown, although the positions are not precise and in some cases are inferred from the genome organization. It is not known whether genes 68 to 34 are transcribed from a separate promoter located between genes 68 and 69 or by antitermination of transcripts originating upstream. Putative transcription terminators are also shown, affecting either rightward (t) or leftward (J) transcription. A terminator is presumed to exist between genes 68 and 69 in order to prevent transcription of downstream genes (68 to 34) during lysogeny.

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Figure 3.

Map of the phage L5 genome. The 52,297-bp genome of L5 is shown as the horizontal bar, with vertical bars spaced 1 kb apart; the attachment site (attP) is located near the center of the genome. The shaded boxes represent the putative L5 genes, with those above the genome being transcribed rightward and those below the genome being transcribed leftward; the different shadings and vertical heights depict the reading frame for each gene. The functions of some of the genes are indicated. At the bottom, the locations of putative promoters are shown, although the positions are not precise and in some cases are inferred from the genome organization. It is not known whether genes 68 to 34 are transcribed from a separate promoter located between genes 68 and 69 or by antitermination of transcripts originating upstream. Putative transcription terminators are also shown, affecting either rightward (t) or leftward (J) transcription. A terminator is presumed to exist between genes 68 and 69 in order to prevent transcription of downstream genes (68 to 34) during lysogeny.

Comparison of regions of lambda and L5 left arms. Segments of the L5 and lambda left arms aligned by the 5′ ends of the major head subunit genes (E and 17 in lambda and L5, respectively) are shown; the coordinates of each segment in the whole genome are given below the genes. Note the similarity of the organization of genes with similar functions despite the lack of sequence similarity. In lambda, gpT is not made, but gpG-T is synthesized as a result of a translational frameshift at the 3′ end of the G gene ( ↑ ); genes 24 and 25 of L5 are organized such that gp24-25 could also be made by a related mechanism.

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Figure 4.

Comparison of regions of lambda and L5 left arms. Segments of the L5 and lambda left arms aligned by the 5′ ends of the major head subunit genes (E and 17 in lambda and L5, respectively) are shown; the coordinates of each segment in the whole genome are given below the genes. Note the similarity of the organization of genes with similar functions despite the lack of sequence similarity. In lambda, gpT is not made, but gpG-T is synthesized as a result of a translational frameshift at the 3′ end of the G gene ( ↑ ); genes 24 and 25 of L5 are organized such that gp24-25 could also be made by a related mechanism.

Integration of phage L5. The circular form of the L5 genome (with ligated cohesive ends) is shown at the top. Some notable features, such as the positions of sites cos and attP, the putative promoters Plate, Pimm, and Pint (located upstream of genes 1, 71, and 33, respectively), and the genes 29 to 32, 35 to 38, int, and xis, are indicated. The directions of transcription of the genes are indicated by arrows. At the bottom is shown a segment of the mycobacterial chromosome representing the attB site that overlaps the 3′ end of the tRNAGly gene. Integration of the L5 genome involves a site-specific recombination event between the attP and attB sites, which share a 43-bp sequence, that is catalyzed by integrase (gplnt) and a mycobacterial integration host factor (mlHF); excision requires excisionase (gpXis) in addition to these two proteins. We propose that in the integrated prophage state, a small amount of gplnt is constantly synthesized by the autoregulated promoter Pint and that excision is prevented by tight regulation of gpXis expression. Although a terminator between genes 68 and 69 (Fig. 1) may stop most transcripts from Pimm getting through to xis, a second terminator downstream of gene 35 may further prevent xis expression, thus ensuring the integrated state.

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Figure 5.

Integration of phage L5. The circular form of the L5 genome (with ligated cohesive ends) is shown at the top. Some notable features, such as the positions of sites cos and attP, the putative promoters Plate, Pimm, and Pint (located upstream of genes 1, 71, and 33, respectively), and the genes 29 to 32, 35 to 38, int, and xis, are indicated. The directions of transcription of the genes are indicated by arrows. At the bottom is shown a segment of the mycobacterial chromosome representing the attB site that overlaps the 3′ end of the tRNAGly gene. Integration of the L5 genome involves a site-specific recombination event between the attP and attB sites, which share a 43-bp sequence, that is catalyzed by integrase (gplnt) and a mycobacterial integration host factor (mlHF); excision requires excisionase (gpXis) in addition to these two proteins. We propose that in the integrated prophage state, a small amount of gplnt is constantly synthesized by the autoregulated promoter Pint and that excision is prevented by tight regulation of gpXis expression. Although a terminator between genes 68 and 69 (Fig. 1) may stop most transcripts from Pimm getting through to xis, a second terminator downstream of gene 35 may further prevent xis expression, thus ensuring the integrated state.

Luciferase reporter phage idea. The idea is to introduce into a mycobacteriophage the luciferase gene from fireflies (FF lux) that has been fused to a mycobacterial promoter as a reporter gene. The phage will find mycobacterial cells and inject their DNAs, and the injected luciferase gene will be expressed. Upon addition of the luciferin substrate, the bacteria will glow. Thus, the test has tremendous sensitivity based on the luciferase reaction that yields light and exquisite specificity based on the host specificity of the mycobacteriophage.

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Figure 6.

Luciferase reporter phage idea. The idea is to introduce into a mycobacteriophage the luciferase gene from fireflies (FF lux) that has been fused to a mycobacterial promoter as a reporter gene. The phage will find mycobacterial cells and inject their DNAs, and the injected luciferase gene will be expressed. Upon addition of the luciferin substrate, the bacteria will glow. Thus, the test has tremendous sensitivity based on the luciferase reaction that yields light and exquisite specificity based on the host specificity of the mycobacteriophage.

“Turn on the light assay.” This assay is based on the observation that luciferase reporter phages will make M. tuberculosis cells glow. Light production requires that the phage DNA be injected and replicated and that the luciferase gene be expressed and have sufficient ATP present within the cell. Anything that interferes with any of these steps, such as contact with drugs, will prohibit light production. Drug screening can be performed by dividing the M. tuberculosis cells into aliquots and incubating each aliquot with a different drug. If the strain is drug susceptible, the cell will become sick and no light will be produced. However, if the strain is drug resistant, the M. tuberculosis cells will be unaffected and light will be produced. Thus, in this figure, the strain is resistant to rifampin (RIF) but susceptible to isoniazid (INH), streptomycin (STR), and ethambutol (ETH).

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Figure 7.

“Turn on the light assay.” This assay is based on the observation that luciferase reporter phages will make M. tuberculosis cells glow. Light production requires that the phage DNA be injected and replicated and that the luciferase gene be expressed and have sufficient ATP present within the cell. Anything that interferes with any of these steps, such as contact with drugs, will prohibit light production. Drug screening can be performed by dividing the M. tuberculosis cells into aliquots and incubating each aliquot with a different drug. If the strain is drug susceptible, the cell will become sick and no light will be produced. However, if the strain is drug resistant, the M. tuberculosis cells will be unaffected and light will be produced. Thus, in this figure, the strain is resistant to rifampin (RIF) but susceptible to isoniazid (INH), streptomycin (STR), and ethambutol (ETH).